Preparation of chimaeric antibodies using the recombinant PCR strategy

ABSTRACT

The invention relates to a method of producing a chimaeric antibody in which the CDR of a first antibody is spliced between the framework regions of a second antibody. The method is performed using a template comprising two framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR. Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD. However, the primers B and C also contain, at their 5&#39; ends, additional sequence corresponding to all or at least part of the donor CDR sequence. Primers B and C overlap by a length sufficient to permit annealing of their 5&#39; ends to each other under conditions which allow a polymerase chain reaction to be performed and thereby incorporate all of the donor CDR sequence. The amplified regions AB and CD may undergo splice overlap extension to produce the chimaeric product in a single reaction.

The present invention relates to the preparation of chimaericantibodies. The invention is typically applicable to the production ofhumanised antibodies.

Antibodies typically comprise two heavy chains linked together bydisulphide bonds and two light chains. Each light chain is linked to arespective heavy chain by disulphide bonds. Each heavy chain has at oneend a variable domain followed by a number of constant domains. Eachlight chain has a variable domain at one end and a constant domain atits other end. The light chain variable domain is aligned with thevariable domain of the heavy chain. The light chain constant domain isaligned with the first constant domain of the heavy chain. The constantdomains in the light and heavy chains are not involved directly inbinding the antibody to antigen.

The variable domains of each pair of light and heavy chains form theantigen binding site. The domains on the light and heavy chains have thesame general structure and each domain comprises a framework of fourregions, whose sequences are relatively conserved, connected by threecomplementarily determining regions (CDRs). The four framework regionslargely adopt a beta-sheet conformation and the CDRs form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs are held in close proximity by the framework regions and, withthe CDRs from the other domain, contribute to the formation of theantigen binding site. CDRs and framework regions of antibodies may bedetermined by reference to Kabat et al ("Sequences of proteins ofimmunological interest" U.S. Dept. of Health and Human Services, U.S.Government Printing Office, 1987).

The preparation of an altered antibody in which the CDRs are derivedfrom a different species to the variable domain framework regions isdisclosed in EP-A-0239400. The CDRs may be derived from a rat or mousemonoclonal antibody. The framework of the variable domains, and theconstant domains, of the altered antibody may be derived from a humanantibody. Such a humanised antibody elicits a negligible immune responsewhen administered to a human compared to the immune response mounted bya human against a rat or mouse antibody. Humanised CAMPATH-1 antibody isdisclosed in EP-A-0328404.

The technique of "overlap extension" involves the use of oligonucleotideprimers complementary to a template nucleotide sequence and thepolymerase chain reaction (PCR) to generate DNA fragments havingoverlapping ends. These fragments are combined in a "fusion" reaction inwhich the overlapping ends anneal allowing the 3' overlap of each strandto serve as a primer for the 3' extension of the complementary strand.Ho et Al (Gene, 77, 51-59 (1989)) describe the use of this technique tointroduce specific alterations in a nucleotide sequence by incorporatingnucleotide changes into the overlapping oligo primers. Using thistechnique of site-directed mutagenesis, those variants of the mousemajor histocompatibility complex class-I gene were generated cloned andanalysed.

Horton et a (Gene, 77 61-68 (1989)) describe a technique of genesplicing by overlap extension (SOE). The technique allows the productionof a hybrid length of DNA, AD, by splicing two pieces of DNA, AB and CD,which are produced by a PCR using primers A, B, C and D. At least partof the primers B and C are complementary to each other. The fragments ABand CD produced by PCR are mixed to allow the positive strand of AB toanneal to the negative strand of CD. The overlap between B and C allowsthe two strands to prime extension of each other. Primers A and D areused to prime a PCR reaction of the extended strands.

The above technique was used to splice a portion (CD) of the mouseH-2K^(b) gene between upstream and downstream regions (AB and EFrespectively) of the corresponding upstream and downstream parts of theH-2L^(d) gene. All three fragments, AB, CD and EF were produced by PCR,using primers A to F. The three fragments were joined by two rounds ofSOE, the first one producing a fragment AD (ie. AB-CD) and the secondproducing the product AF (ie. AB-CD-EF).

According to the present invention, a method has now been devised ofproducing a chimaeric antibody in which the CDR of a first antibody isspliced between the framework regions of a second antibody.

In general, the technique of the present invention is performed using atemplate comprising two framework regions, AB and CD, and between them,the CDR which is to be replaced by a donor CDR. Primers A and B are usedto amplify the framework region AB, and primers C and D used to amplifythe framework region CD. However, the primers B and C also contain, attheir 5' ends, additional sequence corresponding to all or at least partof the donor CDR sequence. Primers B and C overlap by a lengthsufficient to permit annealing of their 5' ends to each other underconditions which allow a polymerase chain reaction (PCR) to be performedand thereby incorporate all of the donor CDR sequence. The amplifiedregions AB and CD may undergo SOE to produce the chimaeric product in asingle reaction.

According to one aspect the present invention provides a method forproducing a double- or single-stranded DNA of formula

    5'F1-M-F2 3'

encoding an antibody chain or fragment thereof in which at least one ofthe complementarity determining regions (CDRs) of the variable region ofthe antibody chain is derived from a first mammalian antibody, and theframework of the variable region is derived from a second, differentmammalian antibody, wherein M comprises DNA encoding a CDR of the secondantibody and F1 and F2 encode sequences flanking M, which methodcomprises;

(i) preparing a single- or double-stranded DNA template of the formula

    5'f1-H-f2 3'

wherein H comprises DNA encoding a CDR of a different specificity from Mand f1 and f2 are substantially homologous to F1 and F2 respectively;

(ii) obtaining DNA oligonucleotide primers A, B, C and D wherein

A

comprises a sequence a¹ which has a 5' end corresponding to the 5' endof F1 and which is identical to a corresponding length of the sequenceF1,

is oriented in a 5' to 3' direction towards H;

B consists of the sequence

    5'b.sup.1 -b.sup.2 3'

wherein

b¹ comprises a sequence complementary to a corresponding length of M andhas a 3' end which is complementary to the 5' end of M, and

b² is complementary to a sequence of corresponding length in F1 and hasa 5' end which starts at the nucleotide complementary to the 3' end ofF1;

C consists of the sequence

    5'c.sup.1 -c.sup.2 3'

wherein

c¹ comprises a sequence identical to the corresponding length of M andhas a 3' end which corresponds to the 3' end of M, and

c² is identical to a sequence of corresponding length in F2 and has a 5'end which starts at the nucleotide corresponding to the 5' end of F2;

D

comprises a sequence d¹ which has a 5' end complementary to the 3' endof F2 and which is complementary to a corresponding length of F2, and

is oriented in a 5' to 3' direction towards H;

and wherein b¹ and c¹ overlap by a length sufficient to permit annealingof their 5' ends to each other under conditions which allow a PCR to beperformed;

(iii) performing, in any desired order, PCR reactions with primer pairsA,B and C,D on the template prepared in (i) above; and

(iv) mixing the products obtained in (iii) above and performing a PCRreaction using primers A and D.

The oligonucleotides may be of any convenient size.

Preferably F1 and F2 each encode at least one human antibody frameworkregion and optionally further CDRs. Preferably H encodes a CDR of saidfirst antibody. Preferably M encodes a non-human CDR region, mostpreferably a murine or rodent CDR.

Primers A and D will usually be at least 12, for example at least 15nucleotides, and more usually from 20 to 30 nucleotides in length. Ifdesired primers A and D may contain at least one restrictionendonuclease recognition site within nucleotides of their 5' ends.Primers B and C will usually be at least 20, for example at least 30nucleotides in length. More usually, these primers will be over 40, forexample 45 to 60 nucleotides long. It is generally possible tosynthesise oligonucleotides of up to 200 nucleotides in length.Generally primers A, B, C and D will thus each be from 15 to 200nucleotides in length.

The length of overlap between b¹ and c¹ may depend on a number offactors, including the total length of B and C and the particular basecomposition of the region of the overlap. However, the overlap willusually be at least 12, for example at least 15, nucleotides. According,to one embodiment, the sequences b¹ and c¹ within the primers B and Care the same number of nucleotides in length. In a preferred embodimentof the invention b¹ and c¹ are both the length of M and thus the overlapis also this length.

Usually, the distance between the 3' end of primer A and the 5' end of Hwill be at least 15 nucleotides. More usually, the distance will be thelength of f1 minus the length of A itself. Similarly, the distancebetween the 3' end of D and the region H will also be at least 15nucleotides, and more usually the length of f2 minus the length of Ditself. According to one embodiment the sequences a¹, b², c² and d¹ ofprimers A, B, C and D respectively are each from 15 to 30 nucleotides inlength.

It will be appreciated that the entire sequence of M and the 5' and 3'regions of F1 and F2 will be determined by the sequence of the primersA, B, C and D.

It is therefore considered inappropriate in this situation to refer to"homology" between these primers and any parts of the sequence F1, M orF2. Instead, the term "corresponding length" as used herein means asequence of the same number of nucleotides and with the identical (orcomplementary) sequence.

With reference to step (i) above, the sequences f1 and f2 will besubstantially homologous to F1 and F2 respectively in that the primers Ato D may be used to introduce minor changes to f1 and f2 in the regionsof these primer sequences.

The regions F1 and F2 comprise DNA encoding at least part of theframework regions either side of the CDR M. F1 and F2 may also encoderegions flanking these sequences, for example into and beyond DNAencoding further CDRs.

According to another aspect, the present invention provides anoligonucleotide 30 to 110 nucleotides in length which consists of thesequence:

    5'o.sup.1 -o.sup.2 3'

wherein o¹ comprises at least 15 nucleotides of a sequence of a CDRregion of non-human origin and o² comprises at least 15 nucleotides of aframework region of human origin. This oligonucleotide is suitable foruse as a primer in the process described above.

According to a still further aspect, the present invention provides amethod for producing a double- or single-stranded DNA of formula

    5'F1-M1-F2-M2-F3-M3-F4 3'

encoding an antibody chain or fragment thereof in which the threecomplementarity determining regions (CDRs) of the variable region of theantibody chain are derived from a first mammalian antibody, and the fourframework regions of the variable domain are derived from a second,different mammalian antibody, wherein M1, M2 and M3 comprise DNAencoding CDRs of the second antibody and F1, F2, F3 and F4 compriseframework sequences flanking the CDRs M1, M2 and M3, which methodcomprises;

(i) preparing a single- or double-stranded DNA template of the formula

    5'f1-H1-f2-H2-f3-H3-f4 3'

wherein H1, H2 and H3 comprises DNA encoding CDRs of a differentspecificity from M1, M2 and M3, and f1, f2, f3 and f4 are substantiallyhomologous to F1, F2, F3 and F4 respectively;

(ii) obtaining DNA oligonucleotide primers A, B, C, D, E, F, G and Hwherein

A

comprises a sequence a¹ which has a 5' end corresponding to the 5' endof F1 and which is identical to a corresponding length of the sequenceF1,

is oriented in a 5' to 3' direction towards H1;

B consists of the sequence

    5'b.sup.1 -b.sup.2 3'

wherein

b¹ comprises a sequence complementary to a corresponding length of M1and has a 3' end which is complementary to the 5' end of M1, and

b2 is complementary to a sequence of corresponding length in F1 and hasa 5' end which starts at the nucleotide complementary to the 3' end ofF1;

C consists of the sequence

    5'c.sup.1 -c.sup.2 3'

wherein

c¹ comprises a sequence identical to the corresponding length of M1 andhas a 3' end which corresponds to the 3' end of M1, and

c² is identical to a sequence of corresponding length in F2 and has a 5'end which starts at the nucleotide corresponding to the 5' end F2;

D consists of the sequence

    5'd.sup.1 -d.sup.2 3'

wherein

d¹ comprises a sequence complementary to a corresponding length of M2and has a 3' end which is complementary to the 5' end of M2, and

d² is complementary to a sequence of corresponding length in F2 and hasa 5' end which starts at the nucleotide complementary to the 3' end ofF2;

E consists of the sequence

    5'e-e.sup.2 3'

wherein

e¹ comprises a sequence identical to the corresponding length of M2 andhas a 3' end which corresponds to the 3' end of M2, and

e² is identical to a sequence of corresponding length in F3 and has a 5'end which starts at the nucleotide corresponding to the 5' end F3;

F consists of the sequence

    5'f-f.sup.2 3'

wherein

f¹ comprises a sequence complementary to a corresponding length of M3and has a 3' end which is complementary to the 5' end of M3, and

f² is complementary to a sequence of corresponding length in F3 and hasa 5' end which starts at the nucleotide complementary to the 3' end ofF3;

G consists of the sequence

    5'g.sup.1 -g.sup.2 3'

wherein

g¹ comprises a sequence identical to the corresponding length of M3 andhas a 3' end which corresponds to the 3' end of M3, and

g² is identical to a sequence of corresponding length in F4 and has a 5'end which starts at the nucleotide corresponding to the 5' end F4;

H

comprises a sequence h¹ which has a 5' end complementary to the 3' endof F4 and which is complementary to a corresponding length of F4, and

is oriented in a 5' to 3' direction towards H3;

and wherein the pairs b¹ and c¹, d¹ and e¹, and f¹ and g¹ overlap by alength sufficient to permit annealing of their 5' ends to each otherunder conditions which allow a PCR to be performed;

(iii) performing, in any desired order, PCR reactions with primer pairsA,B; C,D; E,F and G,H on the template prepared in (i) above to obtainDNA fragments AB, CD, EF and GH; and

(iv) splicing the fragments obtained in (iii) above to obtain thedesired DNA.

According to one embodiment, F4 comprises the framework sequenceflanking the CDR M3 and DNA encoding all or part of the constant regionof the antibody chain.

Step (iv) may be performed by:

(iva) mixing fragments AB and CD with primers A and D and performing aPCR to obtain a DNA fragment AD;

(ivb) mixing, before, during or following step (iva) above, fragments EFand GH with primers E and H and performing a PCR to obtain a DNAfragment EH; and

(ivc) mixing fragments AD and EH with primers A and H to obtain thedesired DNA.

Alternatively step (iv) may be performed by:

(iva) mixing fragments CD and EF with primers C and F and performing aPCR to obtain a DNA fragment CF; and EITHER:

(ivb-1) mixing fragments AB and CF with primers A and F and performing aPCR to obtain a DNA fragment AF; and

(ivc-1) mixing fragments AF and GH with primers A and H to obtain thedesired DNA; OR:

(ivb-2) mixing fragments CF and GH with primers C and H and performing aPCR to obtain a DNA fragment CH; and

(ivc-2) mixing fragments AB and CH with primers A and H to obtain thedesired DNA.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process according to the present invention. Thedark box indicates DNA sequence from a murine CDR region which isinserted between framework regions of the CAMPATH antibody, replacingthe original CDR (unshaded box). A, B, C and D indicate the PCR primersused, with half-arrows indicating their 5' to 3' orientation.

FIG. 2 shows in detail the key sequences involved in the processillustrated in FIG. 1 (SEQ ID NO:1 to SEQ ID NO:8).

FIG. 3 is a schematic illustration of how the process of the inventionmay be used to replace all three CDR regions of an antibody.

FIG. 4 illustrates in further detail one configuration of primers whichmay be used in the present invention.

Possible variations in the F1 and F2 DNA regions are apparent bycontrasting the embodiments of the invention illustrated in FIGS. 1 and3.

In FIG. 1, a process according to the invention is illustrated showingthe replacement of a single CDR DNA.

The region F2 in FIG. 1 is between primers "C" and "D", starting at the5' end of c² as defined above to the complement of the 5' end of "D".This region encodes a total of 3 framework regions, 2 CDRs and the wholeheavy chain constant region incorporating a stop codon within primer D.In contrast, the DNA of F1, 5' to the CDR being replaced, contains asingle framework and no CDRs.

In FIG. 3, the DNA between primers "C" and "D" encodes a singleframework region. This is because the process illustrated shows thereplacement of all 3 CDRs of DNA encoding the variable region of anantibody. With this arrangement, it should be noted that primer "D"comprises not only the sequence of d¹ but also additional 5' sequenceencoding part of a second CDR region.

When the DNA encoding all 3 CDRs of an antibody chain is to be replaced,the arrangement of FIG. 3 may be used.

Thus, a first set of 4 primers, "A", "B", "C" and "D" (as defined abovefor A, B, C and D) are used to replace all of a first CDR (CDR1) and atleast part of a second CDR, (CDR2). A second set of primers, "E", "F","G" and "H" (defined as for A, B, C and D respectively) are used toreplace a third CDR (CDR3) and at least part of CDR2. In order to ensurethe replacement of CDR2, primers "D" and "E" must overlap by a lengthsufficient to permit annealing of their 5' ends to each other underconditions which allow a PCR to be performed. In essence, replacement ofCDR2 is accomplished by a set of four primers, "C", "D", "E" and "F",defined as for A, B, C and D respectively.

In the embodiment of the invention illustrated by FIG. 3, fragments ABand CD are annealed to provide fragment AD, and fragments EF and GH arespliced to provide fragment EH. Finally AD is spliced with EH to providefragment AH, encoding a variable region in which all 3 CDRs arereplaced.

Other arrangements by which all 3 CDR DNAs may be replaced in a DNAencoding a variable region using primers "A" to "H" as illustrated inFIG. 3 include performing reactions with primer pairs "A"+"B", "C"+"D","E"+"F" and "G"+"H" as illustrated in FIG. 3(1), splicing fragments CDand EF together to produce a fragment CF, and splicing this fragmentwith either first fragment AB and then GH, or vice versa.

Alternatively, the DNA encoding the 3 CDRs may be replaced sequentially.A first reaction using primers "A", "B", "C" and "H" (as shown in FIG. 3and defined as for primers A to D) may be used to replace CDR1, inaccordance with the present invention. A second set of reactions, usingprimers "A", "E", "D" and "H" (as shown in FIG. 3 and defined as forprimers A to D) replaces CDR2. A final set of reactions, using primers"A", "F", "G" and "H" replaces CDR3.

The primers A and D may also, at their 5' ends contain additionalsequences which represent, for example, restriction endonucleaserecognition sequences not represented in f1 or f2.

The sequences of A and D 5' to a¹ and d¹ will be ignored whenconsidering the degree of homology between f1 and F1, and f2 and F2.Similarly, if F1 and/or F2 are shorter than f1 and/or f2 respectively,the additional sequences of f1/f2 for which F1/F2 have no counterpartwill also be ignored when measuring the degree of homology.

All the primers may contain a number, for example 1 to 10, such as 2 to5 nucleotide mismatches between the f1/f2 sequences and thecorresponding or complementary primer sequences. These mismatches may beused to design desired coding changes in the sequences of F1 and F2 whencompared with f1 and f2.

The process of the invention may be used to produce a chimaeric antibodyor fragment thereof in which any one of the CDR regions are replaced. Itmay also be used to replace any two, or all three CDR regions of anantibody variable region.

The process of the invention may be used to replace the DNA encoding oneor more CDRs of a complete antibody light or heavy chains. Fragments ofDNA encoding at least one CDR region may be used. For example, it ispossible to produce antibody fragments such as Fab, F(ab)₂ or Fvfragments, in which the DNA encoding one or both of the light or heavychains has been subjected to the process of the invention.

DNA encoding framework regions and CDRs of antibodies will often bepresent in a vector, for example an expression vector. In some cases, itwill be necessary or desirable that one or both of the primers A and D(or at least their regions a¹ and d¹) correspond to vector sequences,rather than sequences of one of the framework regions flanking the CDRbeing replaced.

The DNA produced according to the invention may be cloned into anysuitable replication or expression vector and introduced into abacterial, yeast, insect or mammalian cell to produce chimaericantibody. Examples of suitable systems for expression are describedbelow.

The antibody chain may be co-expressed with a complementary antibodychain. At least the framework of the variable region and the or eachconstant region of the complementary chain generally are derived fromthe said second species also. A light chain and a heavy chain may beco-expressed. Either or both-chains may have been prepared by theprocess of the invention. Preferably the CDRs of both chains are derivedfrom the same selected antibody. An antibody comprising both expressedchains can be recovered.

The antibody preferably has the structure of a natural antibody or afragment thereof. The antibody may therefore comprise a completeantibody, a (Fab')₂ fragment, a Fab fragment, a light chain dimer or aheavy chain. The antibody may be an IgG, such as an IgG1, IgG2, IgG3 orIgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may be achimaeric antibody of the type described in WO 86/01533.

A chimaeric antibody according to WO 86/01533 comprises an antigenbinding region and a non-immunoglobulin region. The antigen bindingregion is an antibody light chain variable region or heavy chainvariable region. Typically, the chimaeric antibody comprises both lightand heavy chain variable regions. The non-immunoglobulin region is fusedat its C-terminus to the antigen binding region. The non-immunoglobulinregion is typically a non-immunoglobulin protein and may be an enzymeregion, a region derived from a protein having known bindingspecificity, from a protein toxin or indeed from any protein expressedby a gene. The two regions of the chimaeric antibody may be connectedvia a cleavable linker sequence.

The invention is preferably employed to humanise an antibody, typicallya monoclonal antibody and, for example, a rat or mouse antibody. Theframework and constant regions of the resulting antibody are thereforehuman framework and constant regions whilst the CDRs of the light and/orheavy chain of the antibody are rat or mouse CDRs. Preferably all CDRsare rat or mouse CDRs. The antibody produced in accordance with thepresent invention may be a human IgG such as IgG1, IgG2, IgG3, IgG4;IgM; IgA; IgE or IgD carrying rat or mouse CDRs.

The process of the invention is carried out in such a way that theresulting chimaeric antibody retains the antigen binding capability ofthe non-human antibody from which the CDR region(s) is/are derived.

The starting antibody is typically an antibody of a selectedspecificity. In order to ensure that this specificity is retained, thevariable region framework of the antibody is preferably the closestvariable region framework of an antibody of another species. By "aboutthe closest" is meant about the most homologous in terms of amino acidsequences. Preferably there is a homology of at least 50% between thetwo variable regions.

There are four general steps to produce a humanised antibody by themethod according to the invention. These are:

(1) determining the nucleotide and predicted amino acid sequence of thestarting antibody light and heavy chain variable regions;

(2) designing the chimaeric antibody, i.e. deciding which antibodyframework region to use during the process;

(3) identifying the oligonucleotides A, B, C, and D and use of theseprimers in a series of PCR reactions to produce DNA encoding thehumanised antibody; and

(4) the transfection of a suitable host cell line with the DNA andexpression of the humanised antibody.

These four steps are explained below in the context of humanising anantibody. However, they may equally well be applied when reshaping to anantibody of a non-human species.

Step 1: Determining the nucleotide and predicted amino acid sequence ofthe antibody light and heavy chain variable regions

To make a chimaeric antibody only the amino acid sequence of antibody'sheavy and light chain variable regions needs to be known. The sequenceof the constant regions is irrelevant because these do not contribute tothe humanising strategy. The simplest method of determining the variableregion amino acid sequence of an antibody is from cloned cDNA encodingthe heavy and light chain variable region.

There are two general methods for cloning heavy and light chain variableregion cDNAs of a given antibody: (1) via a conventional cDNA library,or (2) via PCR. Both of these methods are widely known. Given thenucleotide sequence of the cDNAs, it is a simple matter to translatethis information into the predicted amino acid sequence of the antibodyvariable regions.

Step 2: Designing the chimaeric antibody

There are several factors to consider in deciding which human antibodysequence to use during the humanisation. The humanisation of light andheavy chains are considered independently of one another, but thereasoning is basically similar for each.

This selection process is based on the following rationale: A givenantibody's antigen specificity and affinity is primarily determined bythe amino acid sequence of the variable region CDRs. Variable regionframework residues have little or no direct contribution. The primaryfunction of the framework regions is to hold the CDRs in their properspacial orientation to recognize antigen. Thus the substitution ofrodent CDRs into a human variable region framework is most likely toresult in retention of their correct spacial orientation if the humanvariable region is highly homologous to the rodent variable region fromwhich they originated. A human variable region should preferably bechosen therefore that is highly homologous to the rodent variableregion(s).

A suitable human antibody variable region sequence can be selected asfollows:

1. Using a computer program, search all available protein (and DNA)databases for those human antibody variable region sequences that aremost homologous to the rodent antibody variable regions. The output of asuitable program is a list of sequences most homologous to the rodentantibody, the percent homology to each sequence, and an alignment ofeach sequence to the rodent sequence. This is done independently forboth the heavy and light chain variable region sequences. The aboveanalyses are more easily accomplished if only human immunoglobulinsequences are included.

2. List the human antibody variable region sequences and compare forhomology. Primarily the comparison is performed on length of CDRs,except CDR3 of the heavy chain which is quite variable. Human heavychains and Kappa and Lambda light chains are divided into subgroups;Heavy chain 3 subgroups, Kappa chain 4 subgroups, Lambda chain 6subgroups. The CDR sizes within each subgroup are similar but varybetween subgroups. It is usually possible to match a rodent Ab CDR toone of the human subgroups as a first approximation of homology.Antibodies bearing CDRs of similar length are then compared for aminoacid sequence homology, especially within the CDRs, but also in thesurrounding framework regions. The human variable region which contains-he most homologous CDRs is chosen as the framework for humanisation.

Step 3: Identification and use of the oligonucleotides A, B, C and D

The general principles for designing primers for PCR are well known, eg.as described by R. K. Saiki ("The Design and Optimisation of the PCR" in"PCR Technology", Ed H. A. Erlich, Stockton Press, (1989)). In addition,specific factors can be considered for each CDR replacement. Wherenecessary, or desired, the 5' ends of A and/or D may encode part or allof a second and/or third CDR. The primers, A and D, may also include attheir 5' ends restriction enzyme sites. These sites can be designedaccording to the vector which will be used to clone the humanisedantibody from the final PCR reaction. The primers B and C must be longenough to overlap by at least a length sufficient to permit annealing oftheir 5' ends to each other under conditions which allow a PCR to beperformed. This will usually require an overlap of at least 12, andpreferably at least 15 nucleotides. One or more of the four primers maydiffer from their template sequences by one or more nucleotides. Thesedifferences may be used to introduce desired coding changes into theframework regions of the antibody.

The primers are then used in a series of PCR reactions using theappropriate template to generate the DNA encoding the humanisedantibody. PCR reactions may be carried out as described by Saiki et al,Science, 29, 487-491 (1988). At each stage the desired product of thePCR reaction may be purified as necessary, for example using selectivefiltration and if necessary the identity of the product can beestablished, for example by gel electrophoresis.

Stem 4: Transfection and expression of the reshaped antibody

Following the reactions to produce the DNA encoding the chimaericantibody, the DNAs are linked to the appropriate DNA encoding light orheavy chain constant region, cloned into an expression vector, andtransfected into a suitable host cell line, preferably a mammalian cellline. These stems can be carried out in routine fashion. A chimaericantibody may therefore be prepared by a process comprising:

a) preparing a first replicable expression vector including a suitablepromoter operably linked to a DNA sequence which encodes at least avariable region of an Ig heavy or light chain, the variable regioncomprising framework regions from a first antibody and CDRs from asecond antibody of different specificity;

b) if necessary, preparing a second replicable expression vectorincluding a suitable promoter operably linked to a DNA sequence whichencodes at least the variable region of a complementary Ig light orheavy chain respectively;

c) transforming a cell line with the first or both prepared vectors; and

d) culturing said transformed cell line to produce said alteredantibody.

Preferably the DNA sequence in step a) encodes both the variable regionand the or each constant region of the antibody chain. The antibody canbe recovered and purified. The cell line which is transformed to producethe altered antibody may be a Chinese Hamster ovary (CHO) cell line oran immortalised mammalian cell line, which is advantageously of lymphoidorigin, such as a myeloma, hybridoma, trioma or quadroma cell line. Thecell line may also comprise a normal lymphoid cell, such as a B-cell,which has been immortalised by transformation with a virus, such as theEpstein-Barr virus. Most preferably, the immortalised cell line is amyeloma cell line or a derivative thereof.

Although the cell line used to produce the chimaeric antibody ispreferably a mammalian cell line, any other suitable cell line, such asa bacterial cell line or a yeast cell line, may alternatively be used.In particular, it is envisaged that E. coli--derived bacterial strainscould be used.

It is known that some immortalised lymphoid cell lines, such as myelomacell lines, in their normal state secrete isolated Ig light or heavychains. If such a cell line is transformed with the vector prepared instep (a) it will not be necessary to carry out step (b) of the process,provided that the normally secreted chain is complementary to thevariable region of the Ig chain encoded by the vector prepared in step(a).

However, where the immortalised cell line does not secrete or does notsecrete a complementary chain, it will be necessary to carry out step(b) This step may be carried out by further manipulating the vectorproduced in step (a) so that this vector encodes not only the variableregion of a chimaeric antibody light or heavy chain, but also thecomplementary variable region.

Alternatively, step (b) is carried out by preparing a second vectorwhich is used to transform the immortalised cell line. This alternativeleads to easier construct preparation, but may not be as preferred asthe first alternative in that production of antibody may be lessefficient.

In the case where the immortalised cell line secretes a complementarylight or heavy chain, the transformed cell line may be produced forexample by transforming a suitable bacteria cell with the vector andthen fusing the bacterial cell with the immortalised cell line byspheroplast fusion. Alternatively, the DNA may be directly introducedinto the immortalised cell line by electroporation or other suitablemethod.

An antibody is consequently produced in which CDRs of a variable regionof an antibody chain are homologous with the corresponding CDRs of anantibody of a Faust mammalian species and in which the framework of thevariable region and the constant regions of the antibody are homologouswith the corresponding framework and constant regions of an antibody ofa second, different, mammalian species. Typically, all three CDRs of thevariable region of a light or heavy chain are derived from the firstspecies.

The antibody may be an IgG, such as IgG1, IgG2, IgG3 or IgG4 IgM, IgA,IgE or IgD. Alternatively, the antibody may be a chimaeric antibody ofthe type described in WO 86/01533.

The recombinant PCR technique of the present invention should allow thegeneration of fully humanised MAb DNA sequences in only two days usingthree rounds of PCR reactions (FIG. 3). Site-directed mutagenesis (Joneset al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332,323-327 (1988)) and oligonucleotide gene synthesis (Queen et al., Proc.Natl. Acad. Sci. U.S.A., 86, 10029-10033 (1989)) have previously beenused for the humanisation of antibodies. The above method has benefitsover these techniques in that smaller oligonucleotides are required inthe procedure, even to transfer large CDRs such as the 19 amino acidCDRH2 present in a number of human IgG subgroup III heavy chains (Clearyet al., Cell, 44, 97-106 (1986)). For example, as illustrated in FIG. 4,where the primary PCR products are designed to overlap in the middle ofthe CDR by 15 bp, the transfer of a 57 bp CDR onto the appropriate FRrequires oligonucleotides of a maximum of 51 bp, assuming a homology of15 bp corresponding to the FR target sequence (Higuchi, Using PCR toengineer DNA, in "PCR Technology" Ed. H. A. Erlich, Stockton Press(1989)).

The technique of the invention is also advantageous over site-directedmutagenesis in that all operations can be performed upon ds DNA withoutthe need for subcloning between ds and ss vectors, thus decreasing thetime and effort required to generate the humanised product.

The invention is illustrated by the following example.

EXAMPLE 1

(a) Recombinant PCR grafting of DX48 CDRH1 onto a human background

The objective was to graft a heavy chain CDR1 (CDRH1) from a ratanti-digoxin mAb (DX48) onto a human Ig backbone. The template used forthe recombinant PCR was the previously humanised CAMPATH-1H heavy chain(Riechmann et al., Nature, 332, 323-327 (1988)), a human IgGl heavychain with NEW (Saul et al., J. Biol. Chem. 253, 585-597 (1978)) Vregion, which had been re-engineered from genomic into cDNAconfiguration, and had subsequently undergone site-directed mutagenesisto replace CAMPATH-1H CDRH2 and CDRH3 sequences with rat DX48 CDRH2 andCDRH3 yielding HUMDXCH.23 ss template in M13 (SEQ ID NO: 1).

PCR reactions (Saiki et al., Science, 239, 487-491 (1988)) were carriedout using ss HUMDXCH.23 template prepared by the method of Sambrook atal., Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold SpringHarbor Laboratory (1989). The reactions were performed in a programmableheating block (Hybaid) using 25 rounds of temperature cycling (94° C.for 1 min, 50° C. for 2 min, and 72° C. for 3 min) followed by a final10 min step at 72° C. 1 μg of each primer, 50 ng of template and 2.5Units of Taq polymerase (Perkin Elmer Cetus) were used in a final volumeof 100 μl with the reaction buffer as recommended by the manufacturer.Synthetic oligonucleotides were made on a 7500 DNA Synthesizer(Milligen).

The approach used is summarised in FIG. 1. Primers used:

A: SEQ ID NO: 2:

B: SEQ ID NO: 3:

C: SEQ ID NO: 4:

D: SEQ ID NO: 5:

Two PCR reactions were carried out using the primer pairs A and B, and Cwith D respectively. Primers A and D correspond to positive and negativestrand oligonucleotides incorporating the HindIII sites at the 5' and 3'termini of the HUMDXCH.23 insert. FIG. 2 shows the nucleotide sequenceof three regions of the HUMDXCH.23 insert incorporating; the first 42 bpat the 5' end of the insert including the start codon of the CAMPATH-1Hleader sequence; the 3' 27 bp of FRH1, the whole length of CDRH1 and the5' 27 bp of FRH2 from CAMPATH-1H; and the final 27 bp at the 3' terminusof the insert including the stop codon at the end of CAMPATH-1H constantregion (CH3). The sequences are separated by 117 bp and 1206 bprespectively. Primer B possesses negative strand sequence from the 3'end of the CAMPATH-1H FRH1 region (with point mutations to convert the27 and Thr 30 of CAMPATH-1H back to the Ser residues present in the NEWFRH¹) together with CDRH1 sequence of DX48 in place of the CAMPATH-1HCDRH1 (FIG. 2). Primer C is made up of the positive strand sequence ofDX48 CDRH1, complementary to the CDRH1 region of primer B, running intothe 5' end of the Campath-1H FRH2 (FIG. 2). In the first round of the ABand CD PCR reactions the HUMDXCH.23 negative strand is synthesised fromprimers B and D respectively (FIG. 1) in subsequent cycles fragments ABand CD (SEQ ID NO: 6 AND NO: 7 respectively) are amplified (FIGS. 1 and2). The products of the two reactions thus constitute the whole lengthof the HUMDXCH-23 insert but with the point mutations stated above andthe Campath-1H CDRH1 replaced by the CORH1 sequence of DX48. FragmentsAB and CD both possess the DX48 CDRH1 sequence such that on denaturationand reannealing the overlapping sequences can anneal.

Excess primers were removed from the AB and CD PCR reactions byselective filtration on a Centricon 100 (Higuchi et al , Nucl. AcidsRes. , 16, 7351-7367 (1988); Amicon). 50 μl of each reaction was placedinto 2 ml of TE (10 m Tris-HCl pH 8, 0.1 mM EDTA) and mixed in the upperreservoir of the Centricon 100. The manufacturer's protocol was followedusing a 25 min centrifugation in a fixed-angle rotor at 1000×G, and thePCR products recovered in a 40 μl retentate.

10 μl of the Centricon 100 retentate was subjected to a recombinant PCRreaction with primers A and D (FIG. 1) using the same conditions asperformed in the primary PCR reactions above. The positive strand offragment AB and the negative strand of CD contain the complementary DX48CDRH1 sequences at their 3' ends, and in the first PCR cycle can annealand serve as primers for one another. Extension of the overlap producesthe recombinant product fragment AD containing the transplanted DX48CDRH1, and this is amplified by primers A and D in the subsequent roundsof PCR (FIGS. 1 and 2). The remaining strands of fragments AB and CD,which are complementary at their 5' ends, are not able to prime eachother, but can act as templates for primers A and D. These generate moreof the primary PCR products, although these fragments are not amplifiedin an exponential manner due to the absence of primers B and C in thereaction.

Gel-purified PCR products were analysed on an agarose gel containing0.8% Type II: Medium EEO Agarose (Sigma) in 89 mM Tris-borate/2 mM EDTA,and visualised by staining with ethidium bromide. The expected sizes ofthe fragments were as follows: AB, 207 bp; CD, 1285 bp; AD, 1471 bp. Thepredominant band observed in each case was of the expected size,although additional minor bands also appeared in reaction AD.

(b) Cloning and sequencing of the recombinant PCR product

Fragment AD (SEQ ID NO: 8) was gel eluted, digested with HindIII (BRL)and cloned into the HindIII site of pUC-18 (BRL). The nucleotidesequence of a clone containing the recombinant molecule was determinedby plasmid priming following the dideoxy chain-termination method(Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977))according to the Sequenase kit (USB) protocol. The entire 1463 nt insertwas found to be of the correct sequence, no misincorporations havingresulted from the two sets of PCR reactions.

EXAMPLE 2

This objective was the humanisation of YFC51.1.1 rat anti-human -CD18heavy and light chains. The DNA sequence of the variable regions of bothchains had been determined and is shown in

SEQ ID NOS 9 and 10--heavy chain and SEQ ID NOS 11 and 12--light chain.

Using the selection procedure described in Step (2) above, the humanvariable domain frameworks of the NEWM heavy chain and REI light chain(Kabat et al, "Sequences of proteins of immunological interest", U.S.Dept. of Health and Human Services, U.S. Government Printing Office(1987)) were chosen for the humanisation process.

The humanised heavy and light chains were constructed as follows.

(i) Light Chain

Light chain oligonucleotide primers:

A_(L) : SEQ ID NO: 13:

B_(L) : SEQ ID NO: 14:

C_(L) : SEQ ID NO: 15:

D_(L) : SEQ ID NO: 16:

E_(L) : SEQ ID NO: 17:

F_(L) : SEQ ID NO: 18:

G_(L) : SEQ ID NO: 19:

H_(L) : SEQ ID NO: 20:

PCR reactions were performed in a programmable heating block (Hybaid)using 20 rounds of temperature cycling (94° C. for 1 min, 50° C. for 2min, and 72° C. for 3 min) followed by a final 10 min step at 72° C. 1μg of each primer, a specified amount of template, and 2.5 units of Taqpolymerase (Perkin Elmer Cetus) were used in a final volume of 100 μlwith the reaction buffer as recommended by the manufacturer.

The initial template for the PCR was CAMPATH-1H light chain (humanisedCAMPATH-1 on RE1 framework; Page and Sydenham, Biotechnology 9, 64-68,(1991)) Four initial PCR reactions were carried out, with long oftemplate per reaction, using the primer pairs A_(L) with B_(L), C_(L)with D_(L), E_(L) with F_(L), and G_(L) with H_(L) respectively. Theproducts of these PCR reactions, fragments AB_(L), CD_(L), EF_(L) andGH_(L) respectively, were purified using Prep-A-Gene (Bio-Rad) followingthe protocol recommended by the manufacturer. Fragments AB_(L) withCD_(L), and EF_(L) with GH_(L) were combined using a quarter of eachpurified product, and subjected to recombinant PCR reactions withprimers A_(L) plus D_(L), and E_(L) plus H_(L) respectively. Theproducts of these reactions, fragments AD_(L) and EH_(L), were purifiedas above, and a quarter of each combined in a recombinant PCR reactionusing primers A_(L) and H_(L). The final humanised light chainrecombinant PCR product, AH_(L), was cloned into the HindIII site ofpUC-18 (BRL) following the method of Crowe et al. (1991), utilising theHindIII sites in primers A_(L) and H_(L). Plasmid isolates weresequenced by the dideoxy chain termination method, and clones of thecorrect sequence chosen.

(ii) Heavy Chain

Heavy chain oligonucleotide primers:

A_(H) : SEQ ID NO: 21:

B_(H) : SEQ ID NO: 22:

C_(H) : SEQ ID NO: 23:

D_(H) : SEQ ID NO: 24:

E_(H) : SEQ ID NO: 25:

F_(H) : SEQ ID NO: 26:

G_(H) : SEQ ID NO: 27:

H_(H) : SEQ ID NO: 28:

The initial template for the PCR was CAMPATH-1H heavy chain. The rodentCDR's were grafted on to the template using the recombinant PCR methodas described in section (i) but using oligonucleotide primers A_(H) toH_(H). The final PCR, i. e. fragments AD_(H) and EH_(H) with primersA_(H) and H_(H), did not give a high yield of product so a fragment AF,was generated (from fragments AD_(H) and EF_(H)) and used with fragmentEH_(H) in a PCR with primers A_(H) and H_(H). Oligonucleotides A_(H) andH_(H) were designed with HindIII and EcoRI sites respectively to enableinitial cloning of the humanised variable region, and a SpeI site wasintroduced into the NEWM framework 4 (FR4) region of oligonucleotideG_(H) to facilitate subsequent cloning of the variable region with asuitable constant region of choice. The SpeI site was chosen so as notto alter the leucine residue at position 109 (numbering according toKabat et al, ibid) of the humanised heavy chain template. Four out ofthe six human heavy J-minigenes possess a leucine at this position;Kabat et al ibid). Thus the use of the engineered SpeI site should begenerally applicable.

The humanised heavy chain variable region recombinant PCR product wascloned into HindIII/EcoRI-cut pUC-18 (BRL), and plasmid isolates of thecorrect sequence were chosen. The FR4 and γ1 constant regions ofCAMPATH-1H heavy chain were PCR cloned into pUC-18 (BRL) usingoligonucleotide primers XH (SEQ ID NO: 29) and YH (SEQ ID NO: 30).Primer X_(H) contains SpeI and HindIII sites, and Y_(H) an EcoRI site.The HINDIII and EcoRI sites were used to clone the PCR product intopUC-18, and plasmid isolates of the correct sequence were selected. Thecomplete heavy chain was subsequently reconstituted from the humanisedvariable region and γ1 constant region clones using the engineered FR4Spel site.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 31    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1457 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 156..182    (D) OTHER INFORMATION: CAMPATH 1H FRH1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 183..197    (D) OTHER INFORMATION: CAMPATH 1H CDRH1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 198..224    (D) OTHER INFORMATION: CAMPATH 1H FRH2    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AAGCTTTACAGTTACTGAGCACACAGGACCTCACCATGNNNNNNNNNNNNNNNNNNNNNN60    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN120    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGCACCGTGTCTGGCTTCACCTTCA180    CCGATTTCTACATGAACTGGGTGAGACAGCCACCTGGACGAGGTNNNNNNNNNNNNNNNN240    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN300    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN360    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN420    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN480    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN540    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN600    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN660    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN720    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN780    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN840    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN900    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN960    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1020    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1080    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1140    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1200    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1260    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1320    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1380    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGGGTAAAT1440    GAGTGCGACGGAAGCTT1457    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GATCAAGCTTTACAGTTACTGAGC24    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GCCCACACCCATACCATAAGTGCTGAAGGTGCTGCCAGACACGGT45    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ACTTATGGTATGGGTGTGGGCTGGGTGAGACAGCCACCTGGACGA45    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GATCAAGCTTCCGTCGCACTCATTTAC27    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 207 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: dsDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GATCAAGCTTTACAGTTACTGAGCACACAGGACCTCACCATGNNNNNNNNNNNNNNNNNN60    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN120    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGCACCGTGTCTGGCAGCACC180    TTCAGCACTTATGGTATGGGTGTGGGC207    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1285 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: dsDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ACTTATGGTATGGGTGTGGGCTGGGTGAGACAGCCACCTGGACGAGGTNNNNNNNNNNNN60    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN120    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN180    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN240    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN300    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN360    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN420    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN480    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN540    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN600    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN660    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN720    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN780    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN840    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN900    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN960    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1020    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1080    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1140    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1200    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGGGT1260    AAATGAGTGCGACGGAAGCTTGATC1285    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1471 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: dsDNA    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 160..186    (D) OTHER INFORMATION: CAMPATH 1H FRH1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 175..177    (D) OTHER INFORMATION: POINT MUTATION    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 184..186    (D) OTHER INFORMATION: POINT MUTATION    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 187..207    (D) OTHER INFORMATION: DK48 CDRH1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 208..234    (D) OTHER INFORMATION: CAMPATH 1H FRH2    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GATCCAGCTTTACAGTTACTGAGCACACAGGACCTCACCATGNNNNNNNNNNNNNNNNNN60    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN120    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGCACCGTGTCTGGCAGCACC180    TTCAGCACTTATGGTATGGGTGTGGGCTGGGTGAGACAGCCACCTGGACGAGGTNNNNNN240    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN300    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN360    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN420    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN480    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN540    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN600    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN660    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN720    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN780    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN840    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN900    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN960    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1020    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1080    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1140    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1200    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1260    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1320    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1380    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN1440    CCGGGTAAATGAGTGCGACGGAAGCTTGATC1471    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 417 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (vi) ORIGINAL SOURCE:    (a) ORGANISM: Rattus rattus    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..417    (D) OTHER INFORMATION: Heavy chain variable region with    signal sequence. YFC51.1.1    (ix) FEATURE:    (A) NAME/KEY: MISC SIGNAL    (B) LOCATION: 1..57    (D) OTHER INFORMATION: Signal Sequence    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 148..162    (D) OTHER INFORMATION: CDR 1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 205..255    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 352..384    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ATGAAATGCAGCTGGATCAACCTCTTCTTGATGGCACTAGCTTCAGGG48    MetLysCysSerTrpIleAsnLeuPheLeuMetAlaLeuAlaSerGly    151015    GTCTACGCAGAAGTGCAGCTGCAACAGTCTGGGCCCGAGCTTCGGAGA96    ValTyrAlaGluValGlnLeuGlnGlnSerGlyProGluLeuArgArg    202530    CCTGGGTCCTCAGTCAAGTTGTCTTGTAAGACTTCTGGCTACAGCATT144    ProGlySerSerValLysLeuSerCysLysThrSerGlyTyrSerIle    354045    AAAGATTACCTTCTGCACTGGGTAAAACATAGGCCAGAATACGGCCTG192    LysAspTyrLeuLeuHisTrpValLysHisArgProGluTyrGlyLeu    505560    GAATGGATAGGATGGATTGATCCTGAGGATGGTGGAACAAAGTATGGT240    GluTrpIleGlyTrpIleAspProGluAspGlyGlyThrLysTyrGly    65707580    CAGAAGTTTCAAAGCAGGGCCACACTCACTGCAGATACATCCTCCAAC288    GlnLysPheGlnSerArgAlaThrLeuThrAlaAspThrSerSerAsn    859095    ACAGCCTACATGCAACTCAGCAGCCTGACGTCTGACGACACAGCAACC336    ThrAlaTyrMetGlnLeuSerSerLeuThrSerAspAspThrAlaThr    100105110    TATTTTTGTACTAGAGGCGAATATAGATACAACTCGTGGTTTGATTAC384    TyrPheCysThrArgGlyGluTyrArgTyrAsnSerTrpPheAspTyr    115120125    TGGGGCCAAGGCACTCTGGTCACTGTCTCTTCA417    TrpGlyGlnGlyThrLeuValThrValSerSer    130135    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 139 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetLysCysSerTrpIleAsnLeuPheLeuMetAlaLeuAlaSerGly    151015    ValTyrAlaGluValGlnLeuGlnGlnSerGlyProGluLeuArgArg    202530    ProGlySerSerValLysLeuSerCysLysThrSerGlyTyrSerIle    354045    LysAspTyrLeuLeuHisTrpValLysHisArgProGluTyrGlyLeu    505560    GluTrpIleGlyTrpIleAspProGluAspGlyGlyThrLysTyrGly    65707580    GlnLysPheGlnSerArgAlaThrLeuThrAlaAspThrSerSerAsn    859095    ThrAlaTyrMetGlnLeuSerSerLeuThrSerAspAspThrAlaThr    100105110    TyrPheCysThrArgGlyGluTyrArgTyrAsnSerTrpPheAspTyr    115120125    TrpGlyGlnGlyThrLeuValThrValSerSer    130135    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 375 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (vi) ORIGINAL SOURCE:    (a) ORGANISM: Rattus rattus    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..375    (D) OTHER INFORMATION: Light chain variable region with    signal sequence. YFC51.1.1    (ix) FEATURE:    (A) NAME/KEY: MISC SIGNAL    (B) LOCATION: 1..60    (D) OTHER INFORMATION: Signal Sequence    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 130..162    (D) OTHER INFORMATION: CDR 1    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 208..228    (D) OTHER INFORMATION: CDR 2    (ix) FEATURE:    (A) NAME/KEY: MISC FEATURE    (B) LOCATION: 325..351    (D) OTHER INFORMATION: CDR 3    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ATGAGGGTCCAGGTTCAGTTTCTGGGGCTCCTTCTGCTCTGGACATCA48    MetArgValGlnValGlnPheLeuGlyLeuLeuLeuLeuTrpThrSer    151015    GGTGCCCAGTGTGATGTCCAGATGACCCAGTCTCCGTCTTATCTTGCT96    GlyAlaGlnCysAspValGlnMetThrGlnSerProSerTyrLeuAla    202530    GCGTCTCCTGGAGAAAGTGTTTCCATCAGTTGCAAGGCAAGTAAGAGC144    AlaSerProGlyGluSerValSerIleSerCysLysAlaSerLysSer    354045    ATTAGCAATTATTTAGCCTGGTATCAACAGAAACCTGGGGAAGCAAAT192    IleSerAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyGluAlaAsn    505560    AAACTTCTTGTCTATTATGGGTCAACTTTGCGATCTGGAATTCCATCG240    LysLeuLeuValTyrTyrGlySerThrLeuArgSerGlyIleProSer    65707580    AGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGA288    ArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleArg    859095    AACCTGGAGCCTGCAGATTTTGCAGTCTACTACTGTCAACAGTATTAT336    AsnLeuGluProAlaAspPheAlaValTyrTyrCysGlnGlnTyrTyr    100105110    GAAAGACCGCTCACGTTCGGTTCTGGGACCAAGCTGGAG375    GluArgProLeuThrPheGlySerGlyThrLysLeuGlu    115120125    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    MetArgValGlnValGlnPheLeuGlyLeuLeuLeuLeuTrpThrSer    151015    GlyAlaGlnCysAspValGlnMetThrGlnSerProSerTyrLeuAla    202530    AlaSerProGlyGluSerValSerIleSerCysLysAlaSerLysSer    354045    IleSerAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyGluAlaAsn    505560    LysLeuLeuValTyrTyrGlySerThrLeuArgSerGlyIleProSer    65707580    ArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleArg    859095    AsnLeuGluProAlaAspPheAlaValTyrTyrCysGlnGlnTyrTyr    100105110    GluArgProLeuThrPheGlySerGlyThrLysLeuGlu    115120125    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GATCAAGCTTCTCTACAGTTACTGAGCACA30    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GCTAAATAATTGCTAATGCTCTTACTTGCTTTACAGGTGATGG43    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    AGAGCATTAGCAATTATTTAGCCTGGTACCAGCAGAAGCCAGG43    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AGATCGCAAAGTTGACCCATAGTAGATCAGCAGCTTTGGAG41    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    TATGGGTCAACTTTGCGATCTGGTGTGCCAAGCAGATTCAG41    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    CGTGAGCGGTCTTTCATAATACTGTTGGCAGTAGTAGGTGGCGATGT47    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    CAACAGTATTATGAAAGACCGCTCACGTTCGGCCAAGGGACCAAGGT47    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GATCAAGCTTCTAACACTCTCCCCTGTTGA30    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TGGGATCGATCAAGCTTTACAGTTACTGAGC31    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GTGCAGAAGGTAATCGGTGAAGGTGAAGCCAGACAC36    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    GATTACCTTCTGCACTGGGTGAGACAGCCACCTGGA36    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    ATACTTTGTTTCACCATCCTCAGGATCAATCCATCCAATCCACTCAAGACCTCG54    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    GGTGAAACAAAGTATGGTCAGAAGTTTCAAAGCAGAGTGACAATGCTGGTAGAC54    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    CCACGAGTTGTATCTATATTCGCCTCTTGCACAATAATAGACCGC45    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    AGATACAACTCGTGGTTTGATTACTGGGGTCAAGGCTCACTAGTCACAGTCTCC54    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    TAGAGTCCTGAGGGAATTCGGACAGCCGGGAAGGTG36    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GCTGCTCCTTTTAAGCTTTGGGGTCAAGGCTCACTAGTCACAGTCTCC48    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    AAGCTTCCGTCGAATTCATTTACCCGGAGACAG33    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: ssDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    ORIGANISM: Rattus rattus    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    AGTGGATAGACAGATGGGGC20    __________________________________________________________________________

We claim:
 1. A method for producing a double- or single-stranded DNA offormula

    5'F1-M-F2 3'

encoding an antibody chain or fragment thereof in which of thecomplementarity determining regions (CDRs) of the variable region of theantibody chain is derived from a first mammalian antibody, and theframework of the variable region is derived from a second, differentmammalian antibody, wherein M comprises DNA encoding a CDR of the secondantibody and F1 and F2 respectively encode 5' and 3' sequences flankingM, which method comprises; (i) preparing a single- or double-strandedDNA template of the formula

    5'f1-H-f2 3'

wherein H comprises DNA encoding a CDR of a different specificity from Mand f1 and f2 are substantially homologous to F1 and F2 respectively;(ii) obtaining DNA oligonucleotide primers A, B, C and D whereinAcomprises a sequence a¹ which has a 5' end corresponding to the 5' endof F1 and which is identical to a corresponding length of the sequenceF1, is oriented in a 5' to 3' direction towards H; B consists of thesequence

    5'b.sup.1 -b.sup.2 3'

whereinb¹ comprises a sequence complementary to a corresponding lengthof M and has a 3' end which is complementary to the 5' end of M, and b²is complementary to a sequence of corresponding length in F¹ and has a5' end which starts at the nucleotide complementary to the 3' end of F¹; C consists of the sequence

    5'c.sup.1 -c.sup.2 3'

whereinc¹ comprises a sequence identical to the corresponding length ofM and has a 3' end which corresponds to the 3' end of M, and c² isidentical to a sequence of corresponding length in F2 and has a 5' endwhich starts at the nucleotide corresponding to the 5' end of F2; Dcomprises a sequence d¹ which has a 5' end complementary to the 3' endof F2 and which is complementary to a corresponding length of F2, and isoriented in a 5' to 3' direction towards H; and wherein b¹ and c¹overlap by a length sufficient to permit annealing of their 5' ends toeach other under conditions which allow a polymerase chain reaction(PCR) to be performed; (iii) performing, in any desired order, PCRreactions with primers pairs A,B and C,D on the template prepared in (i)above; and (iv) mixing the products obtained in (iii) above andperforming a PCR reaction using primers A and D.
 2. A method accordingto claim 1 wherein F1 and F2 each encode at least one human antibodyframework region, and optionally further CDRs.
 3. A method according toclaim 1 or 2 wherein H encodes a CDR of the said first antibody.
 4. Amethod according to claim 1 wherein M encodes a non-human CDR region. 5.A method according to claim 4 wherein M encodes a murine or rodent CDR.6. A method according to any claim 1 wherein the primers A and D containat least one restriction endonuclease recognition site within 10nucleotides of their 5' ends.
 7. A method according to claim 1 wherein,in the primers B and C, b¹ and c¹ are the same number of nucleotides inlength.
 8. A method according to claim 1 wherein primers A, B, C and Dare each from 15 to 200 nucleotides in length.
 9. A method according toclaim 8 wherein a¹, b², c² and d¹ of primers A, B, C and D respectivelyare each from 15 to 30 nucleotides in length.